RESUMO
BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Modelos Moleculares , Agonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X3 , Trifosfato de Adenosina/farmacologia , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico , Mutação , Oócitos , Conformação Proteica , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/fisiologia , Xenopus laevisRESUMO
BACKGROUND AND PURPOSE: In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH: Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS: Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS: The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.
Assuntos
Antiparasitários/farmacologia , Ivermectina/farmacologia , Receptores Purinérgicos P2X7/fisiologia , Trifosfato de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Benzoxazóis/metabolismo , Cálcio/fisiologia , Células Cultivadas , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Compostos de Quinolínio/metabolismo , Ratos , Ratos Wistar , Receptores Purinérgicos P2X4/fisiologia , Especificidade da EspécieRESUMO
Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP(4-) at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP(4-) binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.
Assuntos
Ativação do Canal Iônico/fisiologia , Prótons , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Cátions/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Receptores Purinérgicos P2X7 , Xenopus laevisRESUMO
Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.
Assuntos
Cátions Monovalentes/farmacologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cloreto de Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Família Multigênica , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Potássio/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Modelos Químicos , Oócitos/fisiologia , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/química , Animais , Células Cultivadas , Simulação por Computador , Cinética , Receptores Purinérgicos P2X7 , Xenopus laevisRESUMO
The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric alpha1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, alpha1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.
Assuntos
Acetilcisteína/análogos & derivados , Membrana Celular/metabolismo , Glicina/metabolismo , Macrolídeos , Ubiquitina/metabolismo , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Animais , Antibacterianos/farmacologia , Cisteína Endopeptidases , DNA Complementar/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Histidina/química , Concentração de Íons de Hidrogênio , Lisina/química , Lisossomos/metabolismo , Modelos Biológicos , Complexos Multienzimáticos/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , RNA Complementar/metabolismo , XenopusRESUMO
Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.
Assuntos
Benzenossulfonatos/farmacologia , Antagonistas do Receptor Purinérgico P2 , Suramina/análogos & derivados , Animais , Cobaias , Íleo/efeitos dos fármacos , Íleo/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Receptores Purinérgicos P2X , Suramina/farmacologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , Xenopus laevisRESUMO
1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X(7) receptor (hP2X(7)), an N-terminally hexahistidyl-tagged hP2X(7) receptor (His-hP2X(7)), or a truncated His-hP2X(7) receptor (His-hP2X(7)DeltaC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique. 2. The activation time course of the wild-type hP2X(7) receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component. 3. The amplitude of the exponentially activating current component of the wild-type hP2X(7) receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity non-cooperative activation sites with apparent dissociation constants of about 4 and 200 microM free ATP(4-), respectively. 4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 microM. 5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X(7) or His-hP2X(7)DeltaC. 6. Our data indicate that the hP2X(7) receptor possesses at least two types of activation sites, which differ in ATP(4-) sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.
Assuntos
Trifosfato de Adenosina/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/fisiologia , Animais , Cátions Bivalentes/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Condutividade Elétrica , Espaço Extracelular/metabolismo , Feminino , Humanos , Cinética , Magnésio/fisiologia , Oócitos , Concentração Osmolar , Fragmentos de Peptídeos/farmacologia , Agonistas do Receptor Purinérgico P2 , Tempo de Reação , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X7 , Sitios de Sequências Rotuladas , Fatores de Tempo , Xenopus laevisRESUMO
Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.
Assuntos
Linfócitos B/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Oócitos/metabolismo , Fenótipo , Ratos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Xenopus laevisRESUMO
P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).
Assuntos
Trifosfato de Adenosina/metabolismo , Polissacarídeos/metabolismo , Receptores Purinérgicos P2/química , Sequência de Aminoácidos , Animais , Glicosilação , Dados de Sequência Molecular , Polissacarídeos/química , Subunidades Proteicas , Ratos , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-Atividade , Xenopus laevisRESUMO
The suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)) bis(1,3,5-naphthalenetrisul fonic acid) (NF279) was analysed with respect to its potency and P2X receptor subtype selectivity. Two-electrode voltage-clamp measurements were performed with Xenopus laevis oocytes expressing homomultimeric rat P2X(1), P2X(2), P2X(3) and human P2X(4) receptors. For the fast desensitising P2X(1) and P2X(3) receptors, IC(50) values strongly depended on whether oocytes were pre-incubated with NF279 prior to ATP superfusion or exposed to NF279 simultaneously with ATP. With a 10 s pre-incubation period of NF279, IC(50) values of 19 nM and 1.62 microM were obtained for rat P2X(1) and P2X(3), respectively. Without pre-incubation, IC(50) values amounted to 2 microM and 85.5 microM for P2X(1) and P2X(3), respectively. For the non-desensitising rat P2X(2) receptor NF279 appeared to act as a competitive antagonist with an IC(50) value of 0.76 microM and a K(B) value of 0.36 microM, as derived from Schild analysis. P2X(4) receptors were the least sensitive subtypes for NF279 (IC(50)>300 microM). The antagonism was fully reversible at all P2X subtypes analysed. Our results indicate that NF279 is a potent P2X(1) receptor-selective and reversible antagonist.
Assuntos
Antagonistas do Receptor Purinérgico P2 , Suramina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Feminino , Humanos , Antagonistas Purinérgicos , Ratos , Receptores Purinérgicos/fisiologia , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X3 , Receptores Purinérgicos P2X4 , Suramina/farmacologia , Xenopus laevisRESUMO
Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.
Assuntos
Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Ácidos Sulfônicos/farmacologia , Animais , Feminino , Cobaias , Técnicas In Vitro , Masculino , Fosfato de Piridoxal/farmacologia , Ratos , XenopusRESUMO
Among suramin analogues, the properties of P2 receptor subtype blockade and ecto-nucleotidase inhibition appear to be controlled by different structural parameters (Fig. 1 and 2, Table 1; Van Rhee et al., 1994; Beukers et al., 1995; Bültmann et al., 1996; Damer et al., 1998a, 1998b; and this study): the molecular size of the compounds, the position of the sulfonic acid residues in the naphthalene rings, the substitution pattern of the benzoyl moieties and the 3'- or 4'-aminobenzoyl-linkages of the phenyl rings "1" and "2". As a result, compounds with different receptor selectivity profiles were obtained. A maximum in potency at and selectivity for P2X1 receptors is reached in NF279, which is a specific P2 receptor antagonist and the compound with the highest P2X1 vs. P2Y receptor and ecto-nucleotidase selectivity presently available.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Receptores Purinérgicos P2/fisiologia , Suramina/análogos & derivados , Suramina/farmacologia , Trifosfato de Adenosina/farmacocinética , Animais , Contração Isométrica/efeitos dos fármacos , Ligantes , Masculino , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Ratos , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervação , Ducto Deferente/fisiologiaAssuntos
Trifosfato de Adenosina/farmacologia , Adjuvantes Imunológicos/farmacologia , Linfócitos B/fisiologia , Receptores Purinérgicos/fisiologia , Linhagem Celular Transformada , Células Cultivadas , Herpesvirus Humano 4 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Tonsila Palatina/imunologia , Receptores Purinérgicos/efeitos dos fármacosRESUMO
8,8'-(Carbonylbis(imino-4, 1 -phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3, 5-naphthalenetrisulfonic acid) (NF279) antagonized P2X receptor-mediated contractions in rat vas deferens, evoked by alpha,beta-methylene ATP (10 microM; pIC50=5.71) without affecting responses mediated via alpha1A-adrenoceptors, adenosine A1 and A2B receptors, histamine H1, muscarinic M3 and nicotinic receptors. The low inhibitory potency of NF279 on P2Y receptors in guinea-pig taenia coli (pA2=4.10) and at ecto-nucleotidases in folliculated Xenopus laevis oocytes (IC50 > 100 microM) indicates that NF279 is a novel specific and selective P2X receptor antagonist.
Assuntos
Antagonistas do Receptor Purinérgico P2 , Suramina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Colo/efeitos dos fármacos , Cobaias , Técnicas In Vitro , Masculino , Oócitos/efeitos dos fármacos , Ratos , Suramina/farmacologia , Ducto Deferente/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Xenopus laevisRESUMO
UNLABELLED: P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. KEYWORDS: blue native PAGE/cross-linking/P2X receptor/quaternary structure.
Assuntos
Canais Iônicos/química , Receptores Purinérgicos P2/metabolismo , Animais , Reagentes de Ligações Cruzadas , Dimerização , Eletroforese em Gel de Poliacrilamida , Glucosídeos/química , Glicosilação/efeitos dos fármacos , Hexosaminidases/farmacologia , Histidina/genética , Canais Iônicos/metabolismo , Ligantes , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Oócitos/metabolismo , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Receptores Colinérgicos/biossíntese , Receptores Colinérgicos/química , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X3 , Xenopus laevisRESUMO
The functional unit of the Na,K-ATPase consists of a catalytic alpha subunit noncovalently linked with a glycoprotein subunit, beta. Using ouabain binding assays and immunoprecipitation of rodent alpha/beta complexes, we show here that all six possible isozymes between three alpha and two beta isoforms can be formed in Xenopus oocytes. Two isoform-specific differences in alpha/beta interactions are observed: (i) alpha1/beta1 and alpha2/beta2 complexes, in contrast to alpha1/beta2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) beta2 subunits must carry N-glycans to combine with alpha1 but not with alpha2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated beta1 subunit comprising the ectodomain results in assembly with alpha1 and alpha2, but not with alpha3; the beta2 ectodomain combines with alpha2 only. A chimera consisting of 81% and 19% of the alpha1 N terminus and alpha2 C terminus, respectively, behaves like alpha2 and coprecipitates with the beta2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the beta2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase alpha and beta subunits.
Assuntos
Glicoproteínas/metabolismo , Isoenzimas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Polaridade Celular , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Glicoproteínas/ultraestrutura , Isoenzimas/ultraestrutura , Camundongos , Modelos Moleculares , Ouabaína/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , ATPase Trocadora de Sódio-Potássio/ultraestruturaRESUMO
Zebrafish beta 3, a full length cDNA clone encoding a zebrafish Na,K-ATPase beta subunit, was isolated. The protein shares highest homology with the beta 3 subunits of amphibians and mammals, slightly less homology with the beta 2 subunits, and is distinct from the beta 1 subunits. The fish beta subunit co-assembled with alpha subunits to form Na,K-ATPase enzymes when expressed in Xenopus oocytes. Embryonic expression was first detected by whole-mount in situ hybridization between 8-12 hr post-fertilization (hpf) in the head mesoderm. Subsequently, and up to 24 hpf, the mRNA was confined to four dorsal domains in the anterior neural tube. After a transient downregulation during the second day, expression was again conspicuous in the nervous system of 3-day-old larvae. Based on its distribution pattern, the fish beta subunit could be involved in setting up regional identities in the developing fish CNS and in the differentiation of distinct cell types.
Assuntos
Proteínas do Tecido Nervoso/genética , ATPase Trocadora de Sódio-Potássio/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , DNA Complementar/genética , Genes , Hibridização In Situ , Larva , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Oócitos , RNA Mensageiro/biossíntese , Alinhamento de Sequência , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/química , Xenopus laevis , Peixe-Zebra/genéticaRESUMO
To study an endocytotic role of the GTP-binding protein RhoA in Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Gö 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Gö 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.
Assuntos
Toxinas Botulínicas , Proteínas de Ligação ao GTP/fisiologia , Oócitos/fisiologia , Xenopus laevis/fisiologia , ADP Ribose Transferases/metabolismo , Animais , Endocitose/efeitos dos fármacos , Feminino , ATPase Trocadora de Sódio-Potássio/fisiologia , Proteína rhoA de Ligação ao GTPRESUMO
Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.